The present invention is concerned with a process for the semi-quantitative determination of hydroperoxides or substances which react with peroxidatively active substances to liberate hydroperoxides in aqueous fluids and with a chromogenic oxidation indicator/color generation inhibitor system which extends the useful range of test systems employing such reagents.
The determination of glucose in body fluids, such as urine or blood, is of importance not only in the case of diabetic patients who must control their sugar input, but also in those situations in which the detection of disease as a public health measure requires the screening of the urine or blood of large numbers of people. Because early diagnosis and continued control are so important in diabetes, a glucose test, to be of greatest value to the patient in monitoring and controlling the disease, must be conveniently rapid, simple enough to serve the needs of the patient and sensitive enough to reflect meaningful variations in urine or blood glucose.
The use of glucose oxidase, a peroxidatively active substance [POD] and a chromogenic oxidation indicator [IND], which is oxidized upon exposure to hydrogen peroxide in the presence of the peroxidatively active substance for the detection of glucose in urine is known. The system involves the formation of hydrogen peroxide by the action of glucose oxidase on glucose: ##STR1## and the resultant oxidation of the indicator [Ind] to its oxidized state [Ind].sub.ox in the presence of a peroxidase which is visually detectable by a color change: ##STR2##
The test described above can be used in the determination of a series of materials which react with oxygen and an oxidase resulting in the formation of hydrogen peroxide. Thus, the system is useful for the detection of occult blood in various body tissues because of the fact that hemoglobin is such a material. In addition, such a system is useful for the detection of cholesterol, triglycerides and uric acid.
Many indicators in these reagent systems are selected to generate color at wavelengths free from endogenous interferents such as bilirubin and hemoglobin to produce a chromogen of high color intensity or extinction. This is especially true in the case of benzidine based indicators such as benzidine, o-tolidine and o-dianisidine as well as the substituted benzidine chromogenic indicators such as 3,3',5,5'-tetramethylbenzidine, 3,3'-diaminobenzidine and 3,3'-dimethoxybenzidine. This property makes them valuable in the detection of micromolar to nanomolar concentration levels of analytes frequently encountered in clinical chemistry and immunochemistry. Unfortunately, significant problems can be encountered in adapting these oxidative indicator systems to the detection of analytes present in higher concentration, especially in situations where sample dilution is undesirable because of instrument or format limitations. Under these conditions, it is often found that too much color intensity is developed at low analyte concentrations making subsequent higher, and in many cases more clinically significant, analyte concentrations more difficult to detect accurately because of limitations in discerning increasingly darker shades of the produced color. This is especially true in the case of the substituted benzidine indicators mentioned above because these indicators quickly turn from light yellow to dark blue/black when used in the determination of relatively high amounts of glucose in urine.
The above described problems are exemplified in the design of test systems for the dry phase detection of urine glucose where concentrations of 0.1 to 5 g/dL are typically observed. Dry phase test systems for this analyte are designed to be detected both visually and instrumentally using reflectance spectroscopy. As is demonstrated in the subsequent examples, most of the maximum color intensity can be produced at the first clinically significant analyte level of interest, making it difficult to visually detect subsequent analyte levels. In addition, reagents stored under elevated temperature conditions, i.e. subjected to thermal stress, frequently exhibit deteriorated color development resulting in assay imprecision.
Aside from the previously mentioned benzidine based indicators, this problem is also encountered with other chromogenic indicator systems which form blue colors upon oxidation such as guaiacol, reduced (leuco or dihydro) 2,6-dichlorophenolindophenol 2,2'-azineobis-(3-ethylbenzothiazoline-6-sulfonic acid), 1-methylbenzothiazol-2-one hydrazone with 3-N,N-dimethylaminobenzoic acid or N,N-Bis-(2-hydroxyethyl)aniline or related coupler systems.
The literature contains references to the use of certain reagents, e.g. hydrazine, hydrazide and hydroxamic acid derivatives, to inhibit the action of horseradish peroxidase (HRP) by complex formation and, in some cases, covalent binding. Thus, it is disclosed in J. Biol. Chem. 248 (1973), 502 that the association and inhibition of HRP was competitive, i.e. could be decreased by adding hydrogen donors such as phenol, aniline and hydroquinone. In Arch. Biochem. Biophys. 140 (1970), 174 there is disclosed a procedure in which the color resulting from the HRP catalyzed reaction of H.sub.2 O.sub.2 with o-dianisidine was used to determine the residual activity of the partially deactivated HRP. In U.S Pat. No. 4,220,713 there is disclosed a diagnostic agent for the detection of hydroperoxides or of substances which react with the liberation of hydroperoxides containing a stabilized oxidation indicator wherein the stabilizer is a 1-arylsemicarbazide of the formula: EQU Ar--NH--NH--CO--NH.sub.2
in which Ar is aryl or aryl substituted with alkyl, alkoxy or halogen.
Semicarbazides which are substituted at the 1 and 4 positions are known. Thus, 4-methyl-1-phenyl semicarbazide is reported in C.A. 30, 48359, Rec. Trav. Chim. 55, 101-21 (1936) and 4-allyl-1-phenylsemicarbazide is reported in C.A. 69, 14270, Appl. Spectrosc. 22, 167-69 (1968). Likewise, there are reported 4-benzyl-1-phenylsemicarbazide in C.A. 5, 17779, Chem. Ber. 44, 560-83 (1911); 4-phenyl-1-dimethylsemicarbazide in C.A. 60, P2950, Fr. Patent 1,334,586. Another 1,4 disubstituted semicarbazide, 4-phenyl-1-(2 pyridyl)semicarbazide is reported in C.A. 57, 12435, J. Chem. Eng. Data, 12 (4), 612-615 (1967).